Using immunohistochemical techniques in combination with retrograde tracing, we have most recently extended our findings on the coexistence of cholecystokinin (CCK) and dopamine (DA) to include the cat, and have shown that CCK coexists with substance P (SP) in the periaqueductal gray in this species as well. Descending supraspinal rat pathways to the spinal cord have been explored more extensively to include enkephalin, SP, and somatostatin. Evidence that a tritransmitter system SP-5HT-TRH innervates the spinal cord has been explored in more detail by examining the next step in the pathway. Dye injections into the diaphragm of the cat combined with immunocytochemistry has revealed that the ventral motor neurons are innervated by these three putative transmitter substances, and studies are presently being planned to explore this innervation physiologically using iontophoretic application of peptides while monitoring motor nucleus activity. In addition, peptide innervation of the paraventricular nucleus of the hypothalamus is being explored preliminary to electrophysilogical studies of the role of CRH an adrenaline on ACTH release. Finally, methodologically we have developed a method for radioimmunocytochemistry which allows us to use autoradiographic techniques to visualize the antibody antigen complex. This permits us to compare putative transmitter innervation areas with autoradiographic analysis of receptor density as well as comparing patterns of immunoreactive peptide terminals which are suspected examples of coexistence.